Chromatography 2015, 2, 691-708; doi:10.

3390/chromatography2040691
OPEN ACCESS

chromatography
ISSN 2227-9075
www.mdpi.com/journal/chromatography
Article

Uncertainty of Blood Alcohol Concentration (BAC) Results as

Related to Instrumental Conditions: Optimization and
Robustness of BAC Analysis Headspace Parameters
Haleigh A. Boswell and Frank L. Dorman *

Department of Biochemistry and Molecular Biology, The Pennsylvania State University, 107 Althouse
Laboratory, University Park, PA 16802, USA

* Author to whom correspondence should be addressed; E-Mail: fld3@psu.edu;

Tel.: +1-814-863-6805; Fax: +1-814-863-8372.

Academic Editor: Andras Guttman

Received: 30 June 2015 / Accepted: 4 December 2015 / Published: 11 December 2015

Abstract: Analysis of blood alcohol concentration is a routine analysis performed in many

forensic laboratories. This analysis commonly utilizes static headspace sampling, followed
by gas chromatography combined with flame ionization detection (GC-FID). Studies have
shown several “optimal” methods for instrumental operating conditions, which are intended
to yield accurate and precise data. Given that different instruments, sampling methods,
application specific columns and parameters are often utilized, it is much less common to
find information on the robustness of these reported conditions. A major problem can arise
when these “optimal” conditions may not also be robust, thus producing data with higher
than desired uncertainty or potentially inaccurate results. The goal of this research was to
incorporate the principles of quality by design (QBD) in the adjustment and determination
of BAC (blood alcohol concentration) instrumental headspace parameters, thereby ensuring
that minor instrumental variations, which occur as a matter of normal work, do not
appreciably affect the final results of this analysis. This study discusses both the QBD
principles as well as the results of the experiments, which allow for determination of more
favorable instrumental headspace conditions. Additionally, method detection limits will also
be reported in order to determine a reporting threshold and the degree of uncertainty at the
common threshold value of 0.08 g/dL. Furthermore, the comparison of two internal
standards, n-propanol and t-butanol, will be investigated. The study showed that an altered
parameter of 85 °C headspace oven temperature and 15 psi headspace vial pressurization
produces the lowest percent relative standard deviation of 1.3% when t-butanol is
Chromatography 2015, 2 692

implemented as an internal standard, at least for one very common platform. The study also
showed that an altered parameter of 100 °C headspace oven temperature and 15-psi
headspace vial pressurization produces the lowest MDL of 0.00002 g/dL when n-propanol
is implemented as an internal standard. These altered headspace parameters have the
potential to produce more precise and accurate BAC determination.

Keywords: blood alcohol concentration; robustness; optimization

1. Introduction

Contrary to popular belief, ethanol is the most commonly abused drug [1]. Despite the significant
decrease in fatality rates over the past several decades due to constant focus by the NTSB (National
Transportation Safety Board), ethanol still prevails in the number of fatalities and injuries each year [2].
Numerous injuries and fatalities are investigated each year resulting from excessive blood alcohol
concentration (BAC) due to consumption of a variety of beverages [3]. This calls for an extremely
reliable and robust analytical method capable of high throughput that can be replicated with precision at
many forensic and toxicological laboratories [4,5]. Strict quality control and assurance are necessary for
this high-volume analysis to achieve an appropriate conviction through the legal system. The common
threshold value that is specified, 0.08 g/dL, has become a specification limit, specifically within the
United States of America [6]. In many cases, the analytical method must determine if the BAC lies
below, at, or above this legal specification limit. Depending on the circumstances, the legal limit may
be specified at 0.02 g/dL for underage drivers or commercial vehicle operators. Additionally, some
situations (military, etc.) may employ a “zero-tolerance” criterion, so true detection limits should be
known so that analytical reports contain “less-than” values, rather than reporting a “0”, a practice used
in some laboratories. At such a low limit, such as 0.02 g/dL, when compared to the common threshold
value of 0.08 g/dL, the analytical method must be sensitive to accurately quantify the BAC.
Headspace gas chromatography (HS-GC) coupled with flame ionization detection (FID) has been the
prevailing technique for BAC due to its simplicity in automation, overall sensitivity, selectivity and
accuracy [5,7]. Dynamic and static headspace are the most common direct gas headspace sampling
methods [8]. Despite dynamic methods being more sensitive than the static methods, it is instrumentally
more complex, and the sensitivity enhancement may not be necessary. Direct injection is another option
for sample introduction into the instrumentation. This method saves money since there is no need for an
expensive sampling instrument. However, continual direct injection, even of dilute blood samples, will lead
to contamination issues within the injector and column and use of more materials such as inlet liners [5].
There is also the consideration of analyte loss with this method [9] and instrument maintenance [4].
These complications will result in a loss of precision and accuracy, and therefore direct injection would
not be ideal, largely based upon the nature of the sample matrix. Another major consideration is the
impractical nature of this method for many laboratories that perform this routine analysis due to the need
to analyze a substantial number of samples daily. Static headspace sampling allows for thermostatic
partitioning of the volatile compounds to occur within a sealed, gas-tight vial [10]. Partitioning occurs
between the sample diluent and the gas phase. The sample is heated and pressurized to allow for inert
Chromatography 2015, 2 693

gas-sampling from the headspace, which is then transferred via a heated transfer line to create a seamless
interface with the GC [11]. Many features such as robust nature, automation and convenience have
resulted in validation of static headspace sampling methods over a wide range of applications [12]. Static
headspace sampling is the ideal method for forensic laboratories preforming BAC analyses when
considering the goals of minimal training, cost savings, time savings and longevity of instrumentation
and materials. This sampling method also provides the best sensitivity with the lowest operational
requirements and is capable of high robustness.
Although the static-HS sampling method and GC-FID analysis are the most utilized techniques for
BAC determination, suboptimal instrumental conditions can affect the reported value. Using an internal
standard method for quantitative analysis helps compensate for matrix differences [13]. Other alcohols
with similar characteristics to ethanol, such as n-propanol and t-butanol, are typically utilized as internal
standards. This allows for a “correction” since the internal standard undergoes the same matrix effects
as the ethanol within the blood, due to their similar chemical properties [14]. Calibration using the internal
standard method typically results in lower percent error when compared to the external standard method.
Taking into consideration the many factors that go into this rather routine analysis, many forensic
laboratories simply follow analytical procedures either provided by the industry or “historical” methods
that have been passed down over the years. This lack of optimization of the instrumental parameters can
lead to an increase in various errors throughout the entire analysis, resulting in higher variance than may
be obtained if all parameters were optimized. Typically, variance is not reported when reporting a BAC
value, and this may lead to additional questions if the data is used in a legal case. In a court of law, if the
variance was known and reported, it may affect the decision in some cases. With limited money and
resources, some laboratories choose to perform this analysis under conditions other than the industry-
suggested parameters (ex. simultaneous dual-column analysis, among others) [15].
The objective of this study was to develop an optimal method that is as robust, accurate and precise
as possible using the most commonly used instrumentation to determine BAC. By systematically altering
the original equipment manufacturer’s (OEM) instrumental headspace parameters (Table 1), a
comparison can be made to determine the optimal parameters necessary for the most accurate and precise
BAC determination. Through the analysis of replicate samples at both 0.08 g/dL and 0.02 g/dL, a degree
of uncertainty at the common threshold value and method detection limits resulting from the modified
instrumental variables were evaluated.

2. Materials and Methods

2.1. Materials

All experiments were conducted using an Agilent Technologies 7890B Series GC equipped with a
split/splitless inlet and dual flame ionization detectors. Two columns, DB-ALC1 (30 m × 0.32 mm ID ×
1.8 µm) and DB-ALC2 (30m × 0.32mm ID × 1.2 µm) were implemented (Agilent Technologies,
Wilmington, DE; cat# 123-9134 and 123-9234, respectively). To employ the advantages of primary and
secondary confirmatory analyses [16,17], a short section of 0.45 mm deactivated silica tubing was
utilized to couple the single injection port to a capillary flow technology, 2-way unpurged splitter
(Agilent Technologies, cat# G3181B). The splitter allows for the single injections made through the
injection port to travel to the two analytical columns, equally. The GC-FID system was also equipped
Chromatography 2015, 2 694

with an Agilent 7697A Headspace Auto-Sampler. A 0.53 mm ID deactivated silica tubing (Agilent
Technologies, cat# 160-2535-5) was utilized within the transfer line in order to connect the automated
headspace sampler oven to the GC injection port. A 0.75-mm i.d. Ultra Inert straight liner was utilized
within the injection port (Agilent Technologies, cat# 5190-4048). Split injections were employed for all
experiments with a split ratio of 20:1. Figure 1 is the instrumental diagram for the overall experimental
setup used.

Table 1. Original Equipment Manufacturer’s (OEM) Static Headspace and GC analysis parameters.
Instrumental GC analysis Parameters
Carrier Gas Helium
Mode Split
Split Ratio 20:1
Inlet
Pressure 19.5 psi
Temperature 250 °C
Split Flow 130.69 mL/min
Temperature 270 °C
H2 Flow 30 mL/min
FID Detectors
Airflow 400 mL/min
Makeup Flow 25 mL/min
Isothermal 40 °C
Oven
Run Time 10.00 min
Instrumental Headspace Parameters
Equilibration Time 7.00 min
Injection 0.50 min
Static Headspace Auto Vial Pressurization 15 psi
sampler Oven Temperature 100 °C
Loop Temperature 110 °C
Transfer Line Temperature 115 °C

Figure 1. Experimental setup using Agilent HS-dual column GC-dual FID for the detection
of blood alcohol.
Chromatography 2015, 2 695

2.2. Analytical Procedure

The first phase in the procedure was to confirm the separation and elution order of a resolution control
standard (Restek, CAT # 36256; Bellefonte, PA, USA) utilizing the original instrumental and headspace
parameters provided by Agilent Technologies. Each compound within the resolution mixture was run
individually to confirm their retention times on both DB-ALC1 and DB-ALC2. These retention times
were then used for comparison against the resolution control standard to ensure there was correct
compound identification. Due to the similar chemical nature of the compounds within the resolution
mixture, co-elutions may occur. Once the desired separation, baseline resolution and Gaussian peak
shape, was confirmed, the principles of quality by design were implemented to determine the robustness
of these “ideal” conditions and conclude if minor instrument variations would impact the overall data
quality. QBD is a systematic approach that involves identifying sources of variability and controlling
manufacturing processes to produce consistent quality over time, and a more thorough discussion of this
subject may be found elsewhere [18].
A combined calibration curve of commercially available and in-house-made standards with ethanol
concentrations ranging from 0.02 g/dL to 0.30 g/dL (Restek, CAT# 36249, 36251, 36260, 36263, 36252,
36253, 36254, and 36255; Bellefonte, PA, USA) was used. The commercially available standards were
prepared by the addition of 500 μL (Hamilton, CAT# 81217; Reno, NV, USA) of each reference standard
ethanol solution to 4.5 mL (VWR CAT# CA89125-306; Bridgeport, NJ, USA) distilled water (diluent)
and 5 μL (SGE CAT# 002000; Austin, TX, USA) diluted internal standard, to obtain a nominal final
volume of 5 mL. The in-house standards were prepared in a similar manner to the commercially available
standards. However, a pre-determined volume of ethanol (200-proof pure ethanol, KOPTEC USP) and
distilled water were used to make stock solutions of the necessary ethanol concentration. n-Propanol
(J.T. Baker, CAT# 9086-01; Center Valley, PA, USA) and t-butanol (J.T. Baker, CAT# 9056-01; Center
Valley, PA, USA) were compared as internal standards for quantification. The internal standard solution
was prepared as a 1:10 (vol/vol) dilution of either n-propanol or t-butanol in distilled water, so that the
final working concentration was 0.0803 g/dL and 0.07809 g/dL, respectively. Stock solutions of each
internal standard were prepared by the addition of 1 mL internal standard and 9 mL of distilled water.
All samples were prepared in flat bottom Polytetrafluoroethylene (PTFE)/silicone 18 mm screw cap
(Agilent Technologies, p/n 5188-2759; Wilmington, DE, USA) 20 mL headspace vials (Agilent
Technologies, p/n 5188-2753; Wilmington, DE, USA). For each new set of headspace parameters, fresh
vials were utilized once and then disposed.
The sample matrix is extremely important when considering the intermolecular interactions that occur
between the solutes and solvent. Using water, as the sample diluent may seem counterintuitive, however,
it has been shown that solubility, or the partition coefficient, in water is decreased, ultimately increasing
the headspace sensitivity. Hachenberg and Schmidt showed that in 100% solvent composition of water,
the overall headspace sensitivity was the greatest, resulting in the greatest peak area of ethanol,
n-propanol, and n-butanol [13]. The solubility of the solutes within a pure water solvent may be
considered an issue, but the alcohols used within this analysis are known to be miscible in water.
The second objective was to evaluate the overall precision and accuracy of the instrumental and
headspace parameters used within the analysis. Ten replicates containing an ethanol concentration of
0.02 g/dL (Restek, CAT# 36249; Bellefonte, PA, USA) were prepared to determine the impact to method
Chromatography 2015, 2 696

detection limits from the various instrumental parameters. Ten additional replicates containing an
ethanol concentration of 0.08 g/dL (Restek, CAT# 36263; Bellefonte, PA, USA) were prepared to
determine the degree of uncertainty at this common threshold value. Both sets of replicates were
prepared in a similar manner to the calibration curve samples using both n-propanol and t-butanol as
internal standards. Continuing calibration verification (CCV) standards at 0.08 g/dL were present at
regular intervals within each sequence in order to evaluate the differences between external and internal
standard quantification, and ensure instrument control throughout the analytical sequence. Each sequence
consisted of a solvent blank (distilled water), eight levels of the calibration curve (0.02 g/dL–0.30 g/dL)
with the respective internal standard (n-propanol or t-butanol), an internal standard blank (internal
standard in distilled water), ten replicates of 0.02 g/dL with the respective internal standard and ten
replicates of 0.08 g/dL with the respective internal standard. A CCV standard of 0.08 g/dL was placed
every 10 samples within the sequence. To ensure there was no cross-contamination, each sample was
prepared in a fresh vial and no vials were re-used. Overall, the sequence consisted of 33 individual
analyses at one set of headspace parameters. Each altered headspace parameter set of oven temperature
and vial pressurization was run twice, one for n-propanol and the other for t-butanol.

2.3. GC Parameters of Instrumentation

Table 1 summarizes the original instrumental parameters for the GC and Headspace Auto-Sampler
provided by Agilent Technologies (original equipment manufacturer, or OEM). Table 2 depicts the
design of experiments (DOE), which includes the several combinations of headspace oven temperature
and headspace vial pressurization that were investigated to determine the robust nature of the original
provided headspace parameters. The oven temperature and vial pressurization within the headspace
auto-sampler were chosen due to their direct effect on the sample. These parameters were of highest
interest, as they would likely have the greatest impact on the resulting data. The overall chromatographic
performance (baseline resolution, Gaussian peak shape, co-elutions and overall baseline) dictated the
varying vial pressurizations that were evaluated at each headspace oven temperature.

Table 2. Headspace parameters evaluated in this study for robustness evaluation.

Headspace Oven Temperature Headspace Vial Pressurization
15 psi
65 °C
30 psi
15 psi
85 °C
30 psi
0 psi
10 psi
100 °C
15 psi
30 psi
15 psi
125 °C
30 psi
135 °C 15 psi
140 °C 15 psi
Chromatography 2015, 2 697

The original combination of 100 °C headspace oven temperature and 15-psi headspace vial
pressurization has consistently shown acceptable accuracy and precision when applied to sterilized
sheep’s blood containing various ethanol concentrations [19]. However, few studies have evaluated
modification of these two specific parameters due to the theory that the headspace temperature must be
lower than that of the sample diluent in order to ensure that little to no sample diluent is vaporized [10].
When comparing the boiling point of water (100 °C) to that of other possible organic solvents such as
Dimethylsulfoxide (DMSO) (189 °C), it is apparent that water requires a lower HS-oven temperature
for a successful analysis. Despite the higher boiling point, these organic solvents can degrade at higher
temperatures. Water was chosen as the diluent for this analysis due to its stable and clean nature [10].
This preparatory method is also consistent with laboratory practices for calibration and calibration
verification. Evaluation of a blood matrix was not performed since the goal was to determine method
performance, not a validation of an actual standard operating procedure (SOP) for a particular laboratory.
Overall, acceptable data in regards to method performance was obtained without the use of blood in
comparison to previous experiments [19], which will be subsequently discussed (Section 3).

2.4. Data Processing and Equations

Agilent OpenLab CDS ChemStation Edition for GC Systems C.01.05 software was used for
instrument control and data processing. Headspace Control software ChemStation Edition B.01.04 was
utilized to control the different variables of the Headspace Auto-sampler.
Both external and internal quantification methods were evaluated. Equation 1 is the general equation
used to perform external standard quantification. The concentration values of the calibration curve were
plotted along the x-axis, while the corresponding ethanol peak areas were plotted along the y-axis [20].
A line of best fit was determined from the scatter points in order to determine the unknown ethanol
concentration (x) of the continuing calibration verification samples. The calculated ethanol concentration
was then compared to the known concentration (0.08 g/dL) and a percent error (Equation 2) was
determined. Internal standard quantification was used for calculating the unknown concentration of the
continuing calibration verification samples. Equation 3 was first used in order to find the Response Factor,
RF, for each concentration of the calibration curve. An average of the response factors (Equation 4) was
taken to give an average relative response factor, RRF, which represents the entire calibration curve.
The RRF value was then utilized in Equation 5 in order to calculate the unknown concentration of the
ethanol within the continuing calibration verification samples. Equation 2 was employed once again in
order to compare the calculated concentration to the actual concentration (0.08 g/dL) with a percent
error. The external and internal quantification methods were calculated for each set of altered headspace
parameters and for each internal standard, n-propanol and t-butanol. The external and internal standard
quantification percent errors demonstrate the overall accuracy of the analysis to determine the ethanol
concentration within a sample.
To evaluate the precision of the analysis, specific calculations were performed using the data collected
from the ten replicate samples at both 0.02 g/dL and 0.08 g/dL. The calculated concentration of each
replicate sample was calculated by using the internal standard quantification method described above.
The same RRF values were used for both 0.02g/dL and 0.08 g/dL since the value represents the entire
calibration curve as a whole. Equation 6 was used to calculate the percent relative standard deviation
that was used in order to determine the precision and repeatability of the blood alcohol concentration
Chromatography 2015, 2 698

analysis at the common threshold value, 0.08 g/dL, and 0.02 g/dL [20]. The standard deviation and mean
were calculated using the function tool within the Excel (Microsoft Corp). Method detection limits
(MDL’s) are essential when determining blood alcohol concentration. This specific measurement
determines the minimum concentration that the analytical method can detect and report with a 99%
confidence. Not only is this measurement qualitatively important, it describes how well the analysis can
be repeated with identical low concentration replicate samples [21]. Equation 7 allows for a MDL
calculation with no limits on the number of replicates. All ten replicates were used within this calculation
that was provided by the EPA [21] at a 99% confidence interval.
Y = mx + b
Y = Ethanol Peak Area
m = slope (1)
x = Unknown Ethanol Concentration
b = y-intercept
| |
% = × 100% (2)
( ℎ )( )
= (3)
( )( ℎ )
= = (4)
ℎ
( ℎ )( ) (5)
=
( )( )

% = = × 100%
̅
= (6)
̅=
MDL = (2.821)(standard deviation) (7)

3. Results and Discussion

In order to ensure the HS-GC-FID was performing as expected, a resolution mixture was analyzed
on both the DB-ALC-1 and DB-ALC-2 columns. The resolution mixture was run at the suggested
instrumental and headspace parameters provided by Agilent Technologies (Table 1). Each component
within the resolution mixture was also run individually to ensure correct identification. Figure 2 depicts
the optimized chromatogram of the resolution mixture on both DB-ALC1 (FID1) and DB-ALC2 (FID2).
It should be noted that the original instrumental temperature program provided by Agilent Technologies
suggested a 10-min run time. In further evaluation of the headspace parameters, the total run time was
changed to 4 min due to ethanol and both internal standards (n-propanol and t-butanol) eluting before
4 min. By shortening the run time for each analysis both time and money are conserved due to less use
of consumables and more runs completing in a typical workday.
Chromatography 2015, 2 699

Figure 2. Gas headspace chromatograms of a resolution mixture on DB-ALC1 (top) and

DB-ALC2 (bottom). The mixture was run under the OEM (Agilent Technologies)
instrumental and headspace parameters (Table 1). All nine components eluted before
4.5 min and 3.5 min, respectively. MEK represents methyl ethyl ketone and ACN represents
acetonitrile. The co-elution within DB-ALC1 (top) was resolved by analyzing individual
compounds. Retention times for the internal standard t-butanol (not shown) are 2.07 min
(DB-ALC1) and 2.18 min (DB-ALC2).

Both internal standards, n-propanol and t-butanol, were also run under the initial OEM instrumental
and headspace parameters on both DB-ALC1 and DB-ALC2 columns. Figure 3 are the chromatograms
from DB-ALC1 and DB-ALC2 (overlapped) of ethanol and n-propanol (internal standard) within the
continuing calibration verification standard. Figure 4 are the chromatograms from DB-ALC1 and
DB-ALC2 (overlapped) of ethanol and t-butanol (internal standard) within the continuing calibration
verification standard. Baseline resolution and acceptable peak shape are displayed for both
chromatograms. There is slight tailing occurring in both n-propanol peaks within Figure 3. Peak tailing
is attributed to reversible adsorption within the transfer line and column. Agilent OpenLab CDS
ChemStation was used for the determination of the peak areas using equivalent integration parameters
in order to maintain consistency.

Figure 3. Gas headspace chromatograms of two continuing calibration verifications

(overlaid) with n-propanol as an internal standard. Samples were run under the original
headspace parameters (Table 1) provided by Agilent Technologies (100 °C headspace oven
temperature, headspace vial pressurization of 15 psi).
Chromatography 2015, 2 700

Figure 4. Gas headspace chromatograms of two continuing calibration verifications

(overlaid) with t-butanol as an internal standard. Samples were run under the OEM
headspace parameters (Table 1) provided by Agilent Technologies (100 °C headspace oven
temperature, headspace vial pressurization of 15 psi).

Each set of altered headspace parameters was evaluated in a single sequence with a full calibration
curve (0.02 g/dL–0.30 g/dL), ten replicates of both 0.02 g/dL and 0.08 g/dL and continuing calibration
verification samples (0.08 g/dL) throughout in order to directly compare the overall results. n-Propanol
and t-butanol were run separately with each set of altered headspace parameters. Both external and
internal quantification methods were performed on all altered headspace parameters; however, the
internal standard method consistently reduced the percent error in the determination of the ethanol
concentration. Table 3 presents all the data collected using n-propanol as an internal standard. Table 4
presents all the collected data using t-butanol as an internal standard.
The comparison of the data collected with n-propanol as an internal standard showed overall lower
percent relative standard deviations at the common threshold of 0.08 g/dL at three different HS-oven
and headspace vial pressurization parameters: (1) 85 °C, 15 psi, (2) 100 °C, 10 psi, and (3) 100 °C, 30 psi.
When compared to the original instrumental parameters provided by Agilent Technologies, these three
parameters produced about 1.0% to 3.0% lower percent relative standard deviation. The condition of
85 °C headspace oven temperature and 15psi headspace vial pressurization produced the overall lowest
percent relative standard deviation of about 1.5%. Figure 5 shows graphically the small variations in
relative standard deviation amongst the several possible headspace parameters. With the exception of
the altered headspace parameter of 140 °C, the alteration of the headspace oven temperature and
headspace vial pressurization within the studied range has a rather small effect on the accuracy and
precision of the determination of blood alcohol concentration when n-propanol is used as an internal standard.
Chromatography 2015, 2 701

Table 3. n-Propanol altered headspace parameters analysis results and statistics.

Headspace
65 °C, 15 psi 65 °C, 30 psi 85 °C, 15 psi 85 °C, 30 psi 100 °C, 0 psi 100 °C, 10 psi 100 °C, 15 psi 100 °C, 30 psi 125 °C, 15 psi 125 °C, 30 psi 135 °C, 15 psi 140 °C, 15 psi
Parameter

ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC
Column
1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2

Ethanol tR 1.399 1.6 1.383 1.59 1.393 1.6 1.394 1.6 1.577 1.81 1.57 1.807 1.399 1.606 1.575 1.805 1.401 1.62 1.396 1.614 1.407 1.7 1.7 1.8

n-propanol tR 2.351 2.888 2.327 2.878 2.343 2.882 2.346 2.901 2.659 3.28 2.65 3.267 2.353 2.899 2.651 3.265 2.359 2.915 2.348 2.905 2.4 3.02 2.56 3.25

CCAL1
5.03 7.35 3.27 3.39 3.29 7.70 1.13 5.58 3.92 4.95 8.76 8.51 0.24 0.59 4.85 4.79 4.62 6.49 3.34 4.65 14.35 2.79 N/A N/A
% Error

CCAL2
3.62 6.74 1.59 0.53 2.13 7.37 8.27 3.77 9.63 13.14 12.11 11.01 3.27 2.49 6.60 5.81 2.40 7.57 2.99 7.61 38.91 5.54 N/A N/A
% Error

CCAL3
3.74 7.05 4.57 3.11 1.45 6.68 3.64 7.26 12.13 18.54 N/A N/A 5.82 4.79 7.56 7.07 2.53 0.42 4.32 7.92 11.03 0.70 N/A N/A
% Error

0.02 %RSD 5.00 5.78 2.02 2.59 2.13 2.49 13.99 8.25 1.90 3.08 6.99 7.31 4.38 4.53 3.81 3.50 4.52 6.39 5.62 9.77 14.32 3.92 38.39 22.77

MDL 0.0027 0.003 0.001 0.001 0.001 0.001 0.0001 0.00004 0.0011 0.0017 0.0041 0.004 0.0027 0.0026 0.00002 0.00002 0.0026 0.0037 0.003 0.005 0.0084 0.0023 0.0264 0.0144

0.08 %RSD 5.26 5.54 4.19 4.36 1.43 1.61 28.16 25.20 3.58 4.31 3.71 3.88 4.47 4.53 3.65 4.09 2.09 11.02 3.36 5.70 7.45 8.72 16.94 36.87

CCAL: continuing calibration verification samples (0.08 g/dL); MDL: method detection limit (g/dL); tR = retention time (minutes); N/A = not applicable.
Chromatography 2015, 2 702

Table 4. t-Butanol altered parameters analysis results and statistics.

Headspace
65 °C, 15 psi 65 °C, 30 psi 85 °C, 15 psi 85 °C, 30 psi 100 °C, 0 psi 100 °C, 10 psi 100 °C, 15 psi 100 °C, 30 psi 125 °C, 15 psi 125 °C, 30 psi 135 °C, 15 psi 140 °C, 15 psi
Parameter

ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC ALC
Column
1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2

Ethanol tR 1.383 1.596 1.395 1.605 1.383 1.599 1.393 1.6 1.577 1.81 1.575 1.808 1.399 1.606 1.575 1.805 1.4 1.6 1.396 1.62 1.6 1.8 1.5 1.8

t-butanol tR 2.041 2.158 2.057 2.158 2.042 2.163 2.057 2.162 2.328 2.451 2.327 2.451 2.066 2.18 2.327 2.448 2.07 2.193 2.065 2.189 2.25 2.5 2.2 2.4

CCAL1
5.48 5.64 56.26 51.95 6.34 7.23 5.51 4.80 8.06 6.37 4.49 4.35 2.39 1.74 1.20 0.14 0.03 1.22 4.45 5.57 22.32 2.20 23.97 12.36
% Error

CCAL2
0.88 1.08 10.2 4.96 7.11 8.58 6.72 0.63 4.11 5.99 8.00 7.16 8.05 7.37 1.60 1.58 3.75 5.37 2.79 6.71 3.84 0.60 N/A N/A
% Error

CCAL3
10.95 10.42 13.72 5.54 12.11 13.23 11.59 2.01 0.92 1.68 2.84 1.91 9.29 9.66 1.89 1.78 0.81 1.73 10.25 18.16 N/A 58.35 N/A 7.52
% Error

0.02 %RSD 5.09 6.02 N/A N/A 3.90 4.02 10.80 4.50 4.27 5.22 4.14 3.87 17.08 4.26 1.68 2.15 7.54 7.75 5.63 5.93 11.28 12.13 95.04 29.72

MDL 0.0025 0.0027 N/A N/A 0.0017 0.0016 0.0001 0.00002 0.0023 0.0026 0.0022 0.0021 0.002 0.0019 0.001 0.0012 0.0044 0.0056 0.0035 0.0033 0.0061 0.0071 0.0274 0.0134

0.08 %RSD 5.97 6.03 15.78 15.45 1.28 1.33 9.53 6.25 1.79 2.23 3.06 3.22 9.29 9.18 3.32 3.36 9.89 9.65 5.01 10.95 31.21 17.35 92.14 31.82

CCAL: continuing calibration verification samples (0.08 g/dL); MDL: method detection limit; tR = retention time (minutes); N/A = not applicable.
Chromatography 2015, 2 703

Headspace Oven Temperature

140

135

125 30 psi

(oC) 100 15 psi

10 psi
85
0 psi
65

0% 10% 20% 30%

% Relative Standard Deviation

Figure 5. Percent relative standard deviation versus headspace parameters using n-propanol
(internal standard). An average percent value was taken between both DB-ALC1 and
DB-ALC2 from the ten replicate samples of 0.08 g/dL.

The comparison of the data collected with t-butanol as an internal standard showed overall lower
percent relative standard deviations at the common threshold of 0.08 g/dL at three different headspace
parameters: (1) 85 °C, 15 psi, (2) 100 °C, 0 psi, and (3) 100 °C, 10 psi. In comparison to the original
instrumental parameters provided by Agilent Technologies, these three parameters produced about 6.0%
to 8.0% lower percent relative standard deviation. Again, the condition of 85 °C headspace oven
temperature and 15 psi for headspace vial pressurization produced the overall lowest percent relative
standard deviation of about 1.3%. Figure 6 graphically shows more variation amongst the percent
relative standard deviation for the different headspace parameters. The effect of altering the headspace
parameters is much greater on the determination of the blood alcohol concentration when t-butanol is
used as an internal standard.
Headspace Oven Temperature

140

135

125 30 psi
(oC)

100 15 psi
10 psi
85
0 psi
65

0% 10% 20% 30% 40% 50% 60% 70%

% Relative Standard Deviation

Figure 6. Percent relative standard deviation versus headspace parameters using t-butanol
(internal standard). An average percent value was taken from both DB-ALC1 and DB-ALC2
from ten replicates at 0.08 g/dL.
Chromatography 2015, 2 704

Relative standard deviation at the common threshold of 0.08 g/dL shows consistently lower
percentages at an altered headspace parameter of 85 °C for the headspace oven temperature and 15 psi
for the headspace vial pressurization when compared to the original headspace parameters (Table 1) for
both n-propanol and t-butanol as internal standards. Despite the headspace oven temperature being either
below or at the internal standards, t-butanol (82 °C) and n-propanol (97 °C) boiling points, this
temperature has a greater effect on the sample diluent. In order to properly vaporize the analytes within
each sample without the sample diluent interfering, the headspace equilibration temperature should be
lower than the boiling point of the sample diluent used [10]. In the case of water as a sample diluent, the
headspace oven temperature must be lower than 100 °C to avoid vaporizing a large amount of water.
If the temperature was set above 100 °C, the equilibration will result in a high vial pressure, potentially
overloading the GC with a large amount of both the sample diluent and analytes. Both Figures 7 and 8
depict individual replicate samples that were run under altered headspace parameters with a headspace
oven temperature above 100 °C with n-propanol and t-butanol internal standards, respectively.

Figure 7. Gas headspace chromatograms of two replicate samples (overlaid) at 0.02 g/dL
with n-propanol as the internal standard. Samples were run under altered headspace
parameters (135 °C headspace oven temperature, headspace vial pressurization of 15 psi).
The first replicate corresponds to the blue (DB-ALC1) and red (DB-ALC2) signals and the
second replicate corresponds to the green (DB-ALC1) and pink (DB-ALC2) signals.

Figure 8. Gas headspace chromatograms of two replicate samples (overlaid) at 0.08 g/dL
with t-butanol as the internal standard. Samples were run under altered headspace parameters
(140 °C headspace oven temperature, headspace vial pressurization of 15 psi). The first
replicate corresponds to the blue (DB-ALC1) and red (DB-ALC2) signals and the second
replicate corresponds to the green (DB-ALC1) and pink (DB-ALC2) signals.
Chromatography 2015, 2 705

Method detection limits play a key role in determining the limit of quantification for a specific method
of analysis. In most cases, and in the case of blood alcohol concentration determination, the lower the
MDL, the better. This value will not only substantiate the sensitivity of the instrument, it should be
reported along with the quantified result for final reporting. The original instrumental and headspace
parameters provided by Agilent Technologies yielded a MDL of about 0.002 g/dL with n-propanol and
t-butanol [19]. Method detection limit was calculated by multiplying the standard deviation by the
student’s t-value for the correct degrees of freedom.
A concentration of 0.02 g/dL was used in order to determine the method detection limit at the different
headspace parameters. This concentration was chosen for the spike level based on the calculated method
detection limits provided by the OEM parameters (Table 1). The spike level used in the determination of the
method detection limit should lie between the calculated MDL and ten times the calculated value [21]. With
previous values of 0.002 g/dL, a spike level of 0.02 g/dL was an appropriate choice, as seen in Table 5.

Table 5. Calculated method detection limit (MDL).

n-Propanol t-Butanol
FID1 FID2 FID1 FID2
Standard Deviation 0.00087 0.00088 0.000703 0.00067
T-Value 2.821 2.821 2.821 2.821
Calculated MDL (g/dL) 0.00245427 0.00248248 0.0019835 0.00189007
10 × Calc. MDL (g/dL) 0.0245427 0.0248248 0.019835 0.0189007
Actual Spike Level (g/dL) 0.020 0.020 0.020 0.020

The comparison of the data collected with n-propanol as an internal standard showed overall lower
MDLs at three different headspace parameters: (1) 100 °C, 30 psi, (2) 85 °C, 30 psi, and (3) 85 °C, 15psi.
In comparison to the original parameters, these altered parameters produced about a 60% to 99%
decrease in the method detection limit. The condition of 100 °C headspace oven temperature and 30 psi
headspace vial pressurization produced the overall lowest MDL of about 0.00002 g/dL. Figure 9
graphically shows the variation in MDLs amongst the several possible headspace parameters. The effect
of altering the headspace parameters is much greater on the MDL than the percent relative standard
deviation when n-propanol is used as an internal standard.

140
Temperature (oC)

135 30 psi
Headspace Oven

125 15 psi
10 psi
100
0 psi
85
65
0.000 0.001 0.002 0.003 0.004 0.005 0.006 0.007 0.008 0.009 0.010 0.011 0.012 0.013 0.014 0.015 0.016 0.017 0.018 0.019 0.020 0.021 0.022

Method Detection Limit (g/dL)

Figure 9. Method detection limits versus headspace parameters using n-propanol (internal
standard). An average value was taken from both DB-ALC1 and DB-ALC2 from ten
replicates at 0.02 g/dL.
Chromatography 2015, 2 706

The comparison of the data collected with t-butanol as an internal standard showed overall lower
MDLs at three different headspace parameters: (1) 85 °C, 30 psi, (2) 100 °C, 30 psi, and (3) 85 °C, 15 psi.
In comparison to the original parameters, these altered parameters produced about a 96 to 100% decrease
in the method detection limit. The condition of 85 °C headspace oven temperature and 30 psi headspace
vial pressurization produced the overall lowest MDL of about 0.00004 g/dL. Figure 10 graphically
shows the relatively small variation in MDLs amongst the several possible headspace parameters. The
effect of altering the headspace parameters is rather small on the MDL when compared to the percent
relative standard deviation when t-butanol is used as an internal standard.

140
Headspace Oven Temperature (oC)

135 30 psi

125 15 psi

100 10 psi

85 0 psi

65

0 0.003 0.006 0.009 0.012 0.015 0.018 0.021 0.024

Method Detection Limit (g/dL)

Figure 10. Method detection limit versus headspace parameter using t-butanol (internal
standard). An average value was taken from both DB-ALC1 and DB-ALC2 from ten
replicates of 0.02 g/dL.

4. Conclusions

This study concludes that an improvement in accuracy and precision for blood alcohol concentration
can be obtained using altered headspace parameters that produce lower percent relative standard
deviations at the common threshold value of 0.08 g/dL and lower method detection limits.
t-Butanol (RSD = 1.3%) produced a slightly lower percent relative standard deviation at 0.08 g/dL
when compared to n-propanol (RSD = 1.5%); however, both performed optimally at an altered
headspace parameter of 85 °C headspace oven temperature and 15 psi headspace vial pressurization,
relative to the recommended settings by the OEM (Table 1). Despite these values, n-propanol was less
affected by the alteration of the headspace parameters in producing accurate and precise blood alcohol
concentrations. n-Propanol excelled in producing the lowest recorded method detection limit at
0.00002 g/dL while at 100 °C headspace oven temperature and 30 psi headspace vial pressurization.
The lowest recorded method detection limit was produced by t-butanol: 0.00004 g/dL at 85 °C headspace
oven temperature and 30-psi headspace vial pressurization. It appears, based on the data in this study,
that alteration of the OEM headspace parameters (Table 1) produces both an improvement in MDL and
an improvement in precision at 0.08 g/dL, but interestingly these do not seem to occur at the same
headspace parameters.
Chromatography 2015, 2 707

The chromatographic performance of the analytes was the main concern in the alteration of the
headspace parameters. Variations in sample preparation were of no interest in this analysis.
Chromatographic resolution and peak shape are vital in determining peak area for quantification of blood
alcohol determination. With minor alterations of few headspace parameters, the accuracy and precision
of the alcohol concentration can be drastically altered. Determining the optimal parameters are of utmost
importance to achieve the most robust and precise analysis. Therefore, it is recommended from these
data that the headspace oven temperature for this instrumentation be set to 85 °C, with a headspace vial
pressurization of 15 psi, for the best overall performance.

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